Anti-influenza viral composition containing bark or stem extract of alnus japonica

ABSTRACT

The present invention relates to an antiviral composition comprising an  Alnus japonica  extract, more specifically, relates to a method for preparing high activated anti-influenza viral composition, which comprises an extract of the bark or stem of  Alnus japonica , and an anti-influenza viral composition comprising the extract. An extract of the bark or stem of  Alnus japonica  according to the present invention has low toxicity to normal cells, while having an excellent antiviral effect even when administered at low concentration and thus the composition comprising the  Alnus japonica  extract can be used effectively in preventing and treating influenza viral infection.

TECHNICAL FIELD

The present invention relates to an antiviral composition comprising anAlnus japonica extract, more specifically, relates to a method forpreparing an extract of the bark or stem of Alnus japonica, which hashigh anti-influenza viral activity, and an anti-influenza viralcomposition comprising the extract.

BACKGROUND ART

Avian influenza virus belongs to the orthomixoviridae family, and causesdamage to poultry such as chicken, turkey. Avian influenza viruses areclassified into 3 types of high-pathogenic, low-pathogenic andnon-pathogenic avian influenza viruses according to the degree ofpathogenicity, among which the high-pathogenic virus is classified as anOIE List A disease by the World Organization for Animal Health (OIE) and“a category 1 domestic animal infectious disease” in Republic of Korea.

Influenza virus is classified as type A, B or C according to theantigenicity of nucleocapsid protein and matrix protein. Moreover,according to the difference of antigen structure of haemagglutinin (HA)and neuraminidase (NA), the HA is classified into 16 subtypes and NA isclassified into 9 subtypes, wherein HA helps host cell receptor binding,and fusion between host cell membrane and viral envelope to cause virusinfection and NA plays an important role when virus buds out through thecell membrane after proliferation. Theoretically, 144 kinds of virussubtypes could exist by the combination of two proteins.

Infection generally occurs by contact with contaminated secretions,furthermore, and it could be spread through the air, in both particleand droplet forms, human feet, feed delivery vehicles, apparatuses andfeces on the surface of eggs etc. Although there are various symptomsaccording to the pathogenicity of infecting virus, generally, they arerespiratory symptoms, diarrhea and a sharp decline in egg productionetc. Moreover, in some cases, cyanosis appears in the head region suchas crest, edema appears on the face, or feathers are ruffled. Mortalityrate also varies from 0% to 100% according to pathogenicity, but sincethe symptoms are similar to those of Newcastle Disease, infectiouslarynogotracheitis, mycoplasma infection and the like, an accuratediagnosis is required.

High pathogenic avian influenza had occurred 23 times from 1959 to 2003throughout the world, most of them were endemic and contained. Theoutbreaks of highly pathogenic avian influenza subtype H5N1 had occurredin Korea in December 2003, occurred in more than 30 countries includingEurope, Africa and most countries in Southeast Asia such as Japan,China, Thailand, Vietnam and Indonesia and thus have become pandemic.Although it is known that humans cannot become infected with avianinfluenza, prevention of avian influenza is of paramount importance topublic health sector since the case of human infection with H5N1 in1997, isolation of H9N2 avian influenza viruses from humans in 1999 inHong Kong and human cases of H7 avian influenza infection in 2004 inCanada. According to a report of the World Health Organization (WHO),(http://www.who.int/csr/disease/avian_influenza/country/cases_table_(—)2006_(—)06_(—)20/en/index.html),it was confirmed that 228 persons had been infected with H5N1 subtypeviruses and 130 persons of them died during the period of 2003 to Jun.20, 2006 in 10 countries. In Korea, since an outbreak of low pathogenicavian influenza by H9N2 subtype viruses had occurred in 1996 and itreoccurred in 1999.

If an avian influenza outbreak occurs, in most countries, the poultryneeds to be disposed of, and countries where avian influenza outbreakshave occurred cannot export poultry products, thus causing swingeingdamages to poultry industry.

Furthermore, when there is a risk of human infection, the damages spreadto the whole industry including the tourism industry and the transportindustry, thus causing astronomical loss.

Natural substance refers to substances which are minimally processedwithout artificial ingredients, and the natural substances classified asGRAS (Generally Recognized As Safe) can be used without restrictions onthe quantity thereof or foods in which the natural substances are to beused. In domestic industry, the natural substances are classified asnatural additives, and used as food additives, and in foreign countries,it has been used as health foods and medical supplies for user's purposewithout extra limitation, because of its excellent functionality.

Meanwhile, Alnus japonica is a deciduous, dicotyledonous tree in theorder Fagales, family Betulaceae, which is commonly called Alnusjaponica tree. They are distributed in Korea, Japan, China, etc., andgrow in marsh conditions, its height is about 20 m and its bark is adeep purplish-brown color. Its winter bud is a long oval shape just likethe shape of an egg turned upside down, which has three lines and apeduncle. The leaves of Alnus japonica grow alternately, and they areoval shaped, egg-shaped (more or less round on both ends, widest at thebottom) or lanceolate. Both sides of a leaf are lustrous and leafmargins are saw-toothed. The flowers of Alnus japonica bloom inMarch˜April, are unisexual, and form a catkin. Staminate spike bearsstaminate flower and each bract subtends 3˜4 flowers. There are fourperianths and four stamens in each flower. Fruit ripens in October and2˜6 fruits are produced. It is long egg-shaped and looks like a pinecone.

Examples of conventional patents relating to Alnus japonica extractsinclude a cosmetic composition containing an Alnus japonica extract(Korean Patent Publication No. 10-2003-0074500) and a method forpreparing a health drink useful for relieving hangovers, which comprisesextracts of Alnus japonica and green tea leaves (Korean PatentPublication No. 10-2006-0023093), etc.

Recently, many research endeavors are taking place to develop anti-viralagents throughout the world. Lamibudine used for the treatment of HIV(Human Immunodeficiency Virus)-1 and hepatitis B, gancyclovir used forthe treatment of symptoms of herpes virus infection, ribavirin which isused mainly for the treatment of symptoms of respiratory syncytial virusinfection but can be used for the treatment of symptoms of various virusinfection when it is an emergency and zanamivir RELENZA™ and oseltamivirTAMIFLU™ which are synthesized artificially as influenza virusneuraminidase inhibitors are all commercially available after gainingapproval. However, use of amantadine and its analogue, rimantadine,which are approved for treatment of influenza virus A, has decreased dueto the appearance of resistant virus and its side effect. Recently,virus resistant to oseltamivir among H5N1 avian influenza virusesappeared, therefore, there is an urgent need to develop variousantiviral agents.

The present inventors have confirmed antiviral activity of methanolextract of Alnus japonica in Korean Patent Registration No. 10-0721703and Korean Patent Registration No. 10-0769050. However, the abovementioned patents have a disadvantage of showing antiviral activity onlywhen the extracts were administered at high concentration and thus thepossible applications thereof are limited.

Therefore, the present inventors have made an extensive effort todevelop a natural substance having a low toxicity to normal cells, whilehaving an excellent effect of inhibiting influenza virus proliferationeven when administered at low concentration, and as a result, confirmedthat an extract obtained by extracting the bark or stem of Alnusjaponica, which is indigenous to Korea, with 80˜90% ethanol at 30˜80°C., has an excellent anti-influenza virus effect, thereby completing thepresent invention.

SUMMARY OF INVENTION

It is a main object of the present invention to provide a method forpreparing an extract of the bark or stem of Alnus japonica, which hashigh anti-influenza viral activity.

It is another object of the present invention to provide a foodcomposition for preventing or improving influenza viral infection, whichcomprises an extract of the bark or stem of Alnus japonica, prepared bythe above method.

It is still another object of the present invention to provide apharmaceutical composition for preventing or treating influenza viralinfection, which comprises an extract of the bark or stem of Alnusjaponica, prepared by the above method.

In order to achieve the above objects, the present invention provides amethod for preparing an extract of the bark or stem of Alnus japonica,which has anti-influenza viral activity, the method comprising the stepsof: (a) extracting the bark or stem of Korean indigenous Alnus japonicawith 80˜100% ethanol at 30˜80° C.; and (b) recovering the resultingextraction solution.

The present invention also provides a food composition for preventing orimproving influenza viral infection, which comprises an extract of thebark or stem of Alnus japonica, prepared by the above method, and asitologically acceptable supplemental additive.

The present invention also provides a pharmaceutical composition forpreventing or treating influenza viral infection, which comprises anextract of the bark or stem of Alnus japonica, prepared by the abovemethod, as an active ingredient.

The present invention also provides use of an extract of the bark orstem of Alnus japonica prepared by the above method for preventing ortreating influenza viral infection.

The present invention also provides a method for preventing or treatinginfluenza viral infection using an extract of the bark or stem of Alnusjaponica prepared by the above method.

In the present invention, the influenza virus is preferably selectedfrom the group consisting of: human influenza virus, swine influenzavirus, equine influenza virus and avian influenza virus. Preferably, theavian influenza virus is KBNP-0028 (KCTC 10866BP).

Other features and examples of the present invention will be furtherclarified from the following detailed description and the appendedclaims.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a method for preparingan extract of the bark or stem of Alnus japonica, which hasanti-influenza viral activity, the method comprising the steps of: (a)extracting the bark or stem of Korean indigenous Alnus japonica with80˜100% ethanol at 30˜80° C.; and (b) recovering the resultingextraction solution.

In one embodiment of the present invention, after the bark or stem ofAlnus japonica was powdered and extracted with water or ethanol by hotwater extraction, cold water extraction, reflux-cooling extraction orultrasonic extraction, then centrifuged, thus obtaining an extract ofthe bark or stem of Alnus japonica.

In the present invention, after a composition containing an extract ofthe bark or stem of Alnus japonica was added to SPF embryonated eggsinfected with avian influenza virus and cultured, the platehemagglutination test was performed, and as a result, it was confirmedthat the composition containing an extract of the bark or stem of Alnusjaponica has excellent anti-influenza viral effect even whenadministered at low concentration.

In another aspect, the present invention relates to a pharmaceuticalcomposition for preventing or treating influenza viral infection(influenza viral disease), which comprises an extract of the bark orstem of Alnus japonica, prepared by the above method, as an activeingredient.

In the present invention, influenza virus is preferably selected fromthe group consisting of: human influenza virus, swine influenza virus,equine influenza virus, and avian influenza virus. More preferably,avian influenza virus is KBNP-0028 (KCTC 10866BP).

The inventive extract of the bark or stem of Alnus japonica is a naturalsubstance and thus has no toxicity, which enables long-termadministration in high dosage as a medical product.

A composition comprising the inventive extract of the bark or stem ofAlnus japonica can be prepared by mixing together with pharmaceuticalagents such as antihistaminic agents, anti-inflammatory analgesicagents, anti-cancer agents and antibiotics, or can be used incombination therewith.

The composition of the present invention in pharmaceutical dosage formsmay be used in the form of pharmaceutically acceptable salts, and alsomay be used alone or in appropriate association, as well as incombination with other pharmaceutically active compounds.

The pharmaceutical composition comprising the inventive extract may beformulated into an oral preparation such as powders, granules, tablets,capsules, suspensions, emulsions, syrups, aerosols etc, an externalpreparation, a suppository and a sterile injectable solution, accordingto the conventional preparation methods. The pharmaceutical compositioncomprising the inventive extract may comprise carriers, excipients anddiluents, and examples of suitable carriers, excipients and diluentsinclude lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,erythritol, maltitol, starch, gum acacia, alginate, gelatin, calciumphosphate, calcium silicate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineraloil.

The composition of the present invention can be formulated into apreparation form, together with the conventional diluents or excipientssuch as fillers, extenders, binders, wetting agents, disintegrants,surfactants etc. A solid preparation for oral administration includestablets, pills, powders, granules, capsules etc, and the solidpreparation is formulated by mixing the extract with at least oneexcipient, for example, starch, calcium carbonate, sucrose, lactose,gelatin and the like. Also, a lubricant such as magnesium stearate andtalc is used in addition to the excipients. A liquid preparation fororal administration includes suspension, zipeprol, emulsion, syrup andthe like, and various excipients, for example, wetting agents, flavoringagents, fragrances, preservatives etc, can be contained thereto inaddition to conventional diluents such as water and liquid paraffin Apreparation for parenteral administration includes sterile aqueoussolution, non-aqueous solution, suspensions, emulsions, a lyophilizedpreparation, and suppositories. Examples of non-aqueous solution andsuspensions include vegetable oil such as propylene glycol, polyethyleneglycol and olive oil, and injectable esters such as ethyloleate and thelike. Base materials of suppositories include witepsol, macrogol, tween60, cacao butter, laurin butter, glycerol gelatine and the like.

Although the dosage of the extract according to the present inventionvaries depending on the weight and condition of a patient, the severityof the disease, the dosage form, administration route, and treatmentperiod, the dosage can be properly determined by a person skilled in theart. However, in order to achieve the desired effect, the inventiveextract is administered at a dose of 0.01-200 mg/kg per day, preferably0.1-100 mg/kg per day. The foregoing doses may be administered as asingle dose or may be divided into multiple doses per day, and the dosesdo not limit the scope of the present invention in any way.

The inventive extract can be administered to mammals including rats,mice, domestic animals, humans and the like via various routes. The modeof administration may include, for example, oral and rectaladministration, or venous, muscular, subcutaneous, endometrium orintracerebroventricular injections.

In another aspect, the present invention provides a food composition forpreventing or improving influenza viral infection, which comprises anextract of the bark or stem of Alnus japonica, prepared by the abovemethod, and a sitologically acceptable supplemental additive.

In an embodiment of this aspect, the influenza virus is preferablyselected from the group consisting of: human influenza virus, swineinfluenza virus, equine influenza virus and avian influenza virus. Morepreferably, the avian influenza virus is KBNP-0028 (KCTC 10866BP).

The composition comprising the inventive extract or pharmaceuticallyacceptable salts thereof can be used as a main ingredient, food additiveand supplement when preparing various functional foods and healthfunctional foods.

In the present invention, the term “functional foods” refers to a foodwhose functionality is improved by adding the inventive extract thereto.Functionality can be broadly divided into physical functionality such assynthetic flavors and natural flavors physiological functionality. Whenthe inventive extract is added to a general food, such as syntheticflavors and natural flavors and physiological functionality thereof willbe improved. Therefore, in the present invention, the food with improvedfunctionality is broadly defined as functional foods.

Aside from the above, the inventive extract may contain variousnutrients, vitamins, minerals (electrolytes), flavors such as syntheticflavors and natural flavors, coloring matters, enhancer (cheese,chocolate, etc.) pectic acid and its salts, alginic acid and its salts,organic acids, protective colloid thickners, pH control agents,stabilizers, preservatives, glycerins, alcohols and carbonating agentsfor carbonated beverage use, etc. In addition, the extract of thepresent invention may contain natural fruit juices, and fruit pulps forthe provision of fruit juice drinks and vegetable drinks. Theseingredients can be used independently or in combination. The proportionof these additives is not so critical, but generally selected from therange of 0.01˜20 parts by weight based on 100 parts by weight of theinventive extract.

EXAMPLES

Hereinafter, the present invention will be described in more detail byexamples. However, it is obvious to a person skilled in the art thatthese examples are for illustrative purpose only and are not construedto limit the scope of the present invention.

Example 1 Preparation of Alnus japonica Extract 1-1: Preparation ofExtraction Solvent and Extraction at Various Temperatures

The bark of Korean indigenous Alnus japonica purchased from Kyungdongmarket, Seoul, Korea was dried at room temperature for 24 hrs, finelychopped and pulverized. 1 kg of the obtained bark fragments wereextracted with 10 L of 95% ethanol under reflux for 8 hrs at 40° C., 60°C. and 80° C. or extracted with 10 L water under reflux for 4 hrs at100° C., and filtered under vacuum to collect supernatant, followed byeluting useful substances from the obtained fragments. The eluted usefulsubstances are dried under vacuum for 24 hrs to obtain 100 g of Alnusjaponica powder, and the obtained powder was dissolved in 99.9% dimethylsulfoxide (DMSO) solution to a concentration of 20 mg/ml, then used forthe following experiments.

For comparison experiments, Alnus japonica bark extracts prepared by themethod in example 1 of Korean Patent Registration No. 10-0721703 andKorean Patent Registration No. 10-0769050 (an antiviral compositioncomprising Alnus japonica extracts), as a control group.

1-2: Preparation of Alnus japonica Extracts Using Alnus japonica fromDifferent Origins

The bark and stem of Korean indigenous Alnus japonica purchased fromKyungdong market, Seoul, Korea and the bark of Chinese native Alnusjaponica purchased from Yanbian market in China were dried at roomtemperature for 24 hrs, finely chopped and pulverized. 1 kg of theobtained Alnus japonica fragments were extracted with 10 L of 80%ethanol and 95% ethanol under reflux at 40° C., respectively, andfiltered under vacuum to collect supernatant, followed by eluting usefulsubstances from the obtained fragments. The eluted useful substances aredried under vacuum for 24 hrs to obtain 100 g of Alnus japonica powderwas obtained, and the obtained powder was dissolved in 99.9% dimethylsulfoxide (DMSO) solution to a concentration of 20 mg/ml, then used forthe following experiments.

Example 2 Examination of Anti-Viral Effect of Alnus japonica Extracts2-1: Preparation of KBNP-0028

As avian influenza virus used in the experiment, hyperproliferativeKBNP-0028 (KR 2006-0026591) cloned after subculturingA/chicken/Korea/SNU0028/2000(H9N2) virus (it is isolated in Korea in2000) in chick embryo was used. That is, SNU0028[A/chicken/Korea/SNU0028/2000(H9N2); isolation and report to NationalVeterinary Research and Quarantine Service, May 9, 2005] islow-pathogenic avian influenza virus of H9N2 subtype, and isolated fromchickens showing mortality and egg drop syndrome in a chicken farmlocated in North jeola Province in Jan. 28, 2000. The isolation methodis as follows: after kidney and tracheal samples from infected chickensare dissolved, suspended in phosphate buffer, and filterated with 0.45μm filter paper, each sample is inoculated into three allantoic cavitiesof SPF (Specific Pathogen Free) embryonated egg (Sunrise Co., NY), andcultured at 37° C. to obtain allantoic fluid. 20 μl of the allantoicfluid and 20 μl of 0.1% chicken red blood cells, extracted from achicken hatched from the SPF embryonated egg, are dropped on a glassplate, and mixed to carry out the plate hemagglutination test.

As a result, in all of the allantoic fluids, obtained by inoculating thekidney sample and tracheal sample, hemagglutination occurred. The viruswas identified with RT-PCR using H9N2 specific primer and base sequenceanalysis (Kim Min Chul, Master's Thesis, 2002, Seoul NationalUniversity), and stored at −70° C. Among them, the virus isolated fromtracheal sample was used in the experiment.

In order to select a vaccinia strain having high productivity inembryonated eggs, the SNU0028 was diluted with a phosphate buffersolution to a concentration of 0.05 to 0.5 HAU/ml, and 200 μl of thediluted solution was inoculated into the allantoic cavity of10-11-day-old SPF embryonated eggs (Sunrise Co., NY), then cultured forthree days at 37° C. Every day, the embryonated eggs, which died threedays ago, were discarded through egg examination in the morning andafternoon. The embryonated eggs, which survived for three days, werestored for 12˜24 hrs at 4° C., from which allantoic fluid was harvestedto measure the volume and hemagglutination titer of each egg. Amongthem, allantoic fluid having the most quantity and the highesthemagglutination titer was inoculated into embryonated eggs using thesame method as described above, and subcultured 19 times to selectallantoic fluid whose productivity was increased showing highhemagglutination titer and high yield thereof, and thus, the strain wasnamed KBNP-0028 and deposited in GenBank located Eoeundong, Youseonggu,Daejeon city, Korea on Oct. 26, 2005 (KCTC 10866BP).

2-2: Culturing Embryonated Egg Shell Fragments

The egg shell of 10˜11-day-old SPF embryonated eggs (Sunrise Co., NY)was washed with 70% ethanol and chick embryo and all body fluids wereremoved. The resulting egg shell was cut into pieces about 8 mm long and8 mm wide while maintaining villi and allantois adhered to the innersurface of the egg shell, and each piece was added into a 24-wellculture plate. Culture medium was prepared by (i) mixing 199 medium(GIBCO-BRL, NY, USA) with F10 medium (GIBCO-BRL, NY, USA) at a ratio of1:1, (ii) adding 0.075% of sodium bicarbonate and 100 μg/ml ofgentamicin.

The 10˜11-day-old SPF embryonated eggs (Sunrise Co., NY) were infectedwith virus by adding 100 μl of the crude allantoic fluids, KBNP-0028prepared in Example 2-1, which is 4˜10-fold diluted, to the villi andallantois of embryonated egg shell fragments, and culturing for 30 minat 37° C., and added with 1000 μl of the culture medium, then Alnusjaponica extracts prepared in Example 1-1 and Example 1-2 was added to 6well plates at various concentration, respectively, followed byculturing for 7 days at 37° C.

2-3: Test of Antiviral Effect

Culture broth of said virus-infected fluids cultured for 7 days inExample 2-2, which is added with Alnus japonica extracts at variousconcentrations, was taken to carry out plate hemagglutination test. 25μl of the culture broth and 25 μl of chicken red blood cells (0.1%) weredropped on a glass plate and mixed evenly. Virus proliferation wasdetermined according to whether hemagglutination occurred within 2 minby moving the glass plate right and left, and up and down.

TABLE 1 Alnus japonica Control extracts (μg/ml) virus non-virus Extractsolvent 400 200 100 50 6/6 0/6  80° C., 95% ethanol 0/6 0/6 2/6 5/6  60°C., 95% ethanol 0/6 0/6 2/6 5/6  40° C., 95% ethanol 0/6 0/6 1/6 3/6100° C., water 1/6 4/6 6/6 6/6 99.9% methanol 0/6 2/6 6/6 —

As a result, as shown in Table 1, in the sample added with 100° C. waterextract as a negative control, hemagglutination occurred in one of 6test samples at 400 μg/ml, showing partial antiviral effect, however,hemagglutination activity was shown in all of 6 test samples at 50 μg/mland 100 μg/ml, suggesting that virus proliferation was not inhibited.

On the other hand, in the sample added with the 95% ethanol extract (at80° C.), hemagglutination did not occur in all of 6 test samples at 400μg/ml and 200 μg/ml showing that virus proliferation was completelyinhibited, hemagglutination occurred in two of 6 test samples at 100μg/ml, showing partial antiviral effect, and hemagglutination occurredin five of 6 test samples at 50 μg/ml, showing weak antiviral effect.The sample added with 95% ethanol extract (at 60° C.) showed the samehemagglutination results as those of the 95% ethanol extract (at 80°C.). The 95% ethanol extract (at 40° C.) showed the samehemagglutination as those of the 95% ethanol extracts (at 80° C., 65°C., respectively) at 400 μg/ml and 200 μg/ml, and thus, nohemagglutination activity was shown in all samples, suggesting thatviral proliferation was completely inhibited, and showed partialantiviral effect hemagglutination occurred in one of 6 test samples at100 μg/ml and three of 6 test samples at 50 μg/ml, thus confirming thatthe extract shows high antiviral at various effect even at lowconcentration.

Based on extract concentration of 100 μg/ml, antiviral activity atvarious solvents was in the order: 95% ethanol extract (at 40° C.)>95%ethanol extract (at 80° C.) and 95% ethanol extract (at 60° C.)>waterextract (at 100° C.). Therefore, it was determined that 95% Alnusjaponica extract (at 40° C.) is most suitable as the Alnus japonicaextract to prepare the inventive antiviral composition.

In order to compare antiviral effects according to Alnus japonica withdifferent origins, extracted at 40° C. at which the highest inhibitioneffect on virus proliferation was shown and various extract solvents,the same experiment as described above was performed under conditionsshown in Table 2.

TABLE 2 Raw materials to Alnus japonica Control be extracted extracts(μg/ml) virus non-virus and solvents 100 50 25 12.5 6/6 0/6 the bark ofKorean native 2/6 4/6 6/6 6/6 Alnus japonica 80% ethanol extract thebark of Korean native 1/6 3/6 4/6 5/6 Alnus japonica 95% ethanol extractthe stem of Korean native 3/6 5/6 6/6 6/6 Alnus japonica 80% ethanolextract the stem of Korean native 1/6 3/6 6/6 6/6 Alnus japonica 95%ethanol extract the bark of Chinese native 1/6 4/6 6/6 6/6 Alnusjaponica 80% ethanol extract the bark of Chinese native 1/6 5/6 6/6 6/6Alnus japonica 95% ethanol extract

As a result, as shown in Table 2, it was observed that 80% and 95%ethanol extracts of the bark of Korean indigenous Alnus japonica showedexcellent antiviral activity, compared to 80% and 95% ethanol extractsof the stem of Korean indigenous Alnus japonica. Thus, it could beconfirmed that the bark was suitable for use as Alnus japonica extractfor the inventive antiviral composition.

When comparing Alnus japonica from different origins, 80% ethanolextracts of the bark of Korean indigenous Alnus japonica and Chineseindigenous Alnus japonica showed similar activity, and 95% ethanolextract of the bark of Korean native Alnus japonica showed moreexcellent activity than that of 95% ethanol extract of Chinese nativeAlnus japonica, and when comparing antiviral activities at variousextract solvents, 95% ethanol extract showed higher antiviral activitiesthan that of 80% ethanol extract.

From the above the results, it could be confirmed that 95% ethanolextract of the bark of Korean native Alnus japonica extracted at 40° C.shows the most excellent antiviral effect.

Hereinafter, examples of preparations of the pharmaceutical compositioncomprising an Alnus japonica extract according to the present invention,however, these examples are for illustrative purpose only and are notconstrued to limit the scope of the present invention.

Preparation Example 1 Powder Preparation

Extract of Alnus japonica: 20 mg

Lactose: 100 mg Talc: 10 mg

The above ingredients were mixed, and changed in an air-tight pack toprepare a powder preparation

Preparation Example 2 Tablet Preparation

Extract of Alnus japonica: 10 mg

Cornstarch: 100 mg Lactose: 100 mg

Stearin magnesium: 2 mg

The above ingredients were mixed, and tableted according to theconventional method to prepare tablets.

Preparation Example 3 Capsule Preparation

Extract of Alnus japonica: 20 mgCrystalline cellulose: 13.3 mg

Lactose: 65.8 mg

Magnesium stearate: 0.9 mg

The above ingredients were mixed, and changed in a gelatin capsuleaccording to the conventional method to prepare capsules.

Preparation Example 4 Injection Preparation

Extract of Alnus japonica: 10 mg

Mannitol: 180 mg

Sterile distilled water for injection: 2,974 mg

Na₂HPO₄.12H₂O: 26 mg

The above ingredients were added to an ample at the amount shown aboveper ampule (3 ml) according to the conventional preparation method ofinjectable solution.

Preparation Example 5 Liquid Preparation

Extract of Alnus japonica: 20 mgIsomerized sugar: 10 g

Mannitol: 5 g

Proper quantity of purified water

Each ingredient was dissolved in the purified water and added with asuitable amount of lemon flavor to mix the above ingredients, then thepurified water was added to a total volume of 100 ml, followed bysterilizing to change in a brown vial, thus preparing liquidpreparation, according to the conventional liquid preparation method.

INDUSTRIAL APPLICABILITY

As described above in detail, the extract of the bark or stem of Alnusjaponica according to the present invention has low toxicity tochoriollantonic cells which is a normal cell, while having an excellentantiviral effect even when administered at low concentration. Therefore,the composition comprising the Alnus japonica extract can be usedeffectively in preventing and treating influenza viral infection.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof. It is understood that numerous changes andmodifications can be made by those skilled in art without departing fromthe invention concepts disclosed herein.

1. A method for preparing an extract of the bark or stem of Alnusjaponica, which has an anti-influenza viral activity, the methodcomprising the steps of: (a) extracting the bark or stem of Koreanindigenous Alnus japonica with 80˜100% ethanol at 30˜80° C.; and (b)recovering the resulting extraction solution at the same temperature byvacuum concentrating and drying.
 2. A food composition for preventing orimproving influenza viral infection, which comprises an extract of thebark or stem of Alnus japonica, prepared by the method of claim 1, and asitologically acceptable supplemental additive.
 3. The compositionaccording to claim 2, wherein said influenza virus is selected from thegroup consisting of human influenza virus, swine influenza virus, equineinfluenza virus, and avian influenza virus.
 4. The composition accordingto claim 3, wherein said avian influenza virus is KBNP-0028 (KCTC10866BP).
 5. A pharmaceutical composition for preventing or treatinginfluenza viral infection, which comprises an extract of the bark orstem of Alnus japonica, prepared by the method of claim 1, as an activeingredient.
 6. The pharmaceutical composition according to claim 5,wherein said influenza virus is selected from the group consisting ofhuman influenza virus, swine influenza virus, equine influenza virus,and avian influenza virus.
 7. The pharmaceutical composition accordingto claim 6, wherein said avian influenza virus is KBNP-0028 (KCTC10866BP).